In prevention and treatment of diseases, detection of gene mutations including single nucleotide polymorphisms (SNPs) has been carried out widely. For example, the gene of a cancer cell has many mutations, and it is known that these mutations are involved in canceration of the cell. Thus, by detecting the gene mutations in the cell, it is possible to check the possibility of canceration, the stage of cancer progression, and the like, which are considered to be very useful information in treatment. Also, a gene mutation that causes a cancer cell to exhibit a drug resistance has been reported. By detecting this mutation, the effectiveness of a drug to a patient can be determined, which enables more appropriate treatment. For example, regarding chronic myelocytic leukemia (CML) to which medication with an anticancer drug “imatinib” is applied widely, a mutation in the bcr-abl gene (e.g., T315I) is considered to affect the drug resistance. As described above, the detection of gene mutations is useful for early detection and treatment of diseases in the field of clinical practice, so that high reliability of the detection is demanded.
As methods for detecting gene mutations, an ASP (Allele Specific Primer)-PCR (Polymerase Chain Reaction) method (Japanese Patent No. 28538641 and a Tm (Melting temperature) analysis method (Crockett et al., Analytical Biochemistry, 290(1):89-97 (2001)) are known generally. The ASP-PCR method is a method in which PCR is performed using a primer that is complementary to a sequence including a target site and has, in its 3′ region, a base complementary to the base at the target site, thereby amplifying the target sequence including the target site to determine a mutation. For example, in the case where a mutant primer designed so that the target site is a mutant base is used as the primer, the gene examined can be determined as “mutant” if amplification is observed and as “normal” if no amplification is observed. On the other hand, in the case where a normal primer designed so that the target site is a normal base is used as the primer, the gene examined can be determined as “normal” if amplification is observed and as “mutant” if no amplification is observed. In the Tm analysis, for example, first, a target sequence including a target site in a gene is amplified, and thereafter, a hybrid (double-stranded nucleic acid) of the thus-obtained amplification product with a probe that can hybridize to the sequence including the target site is formed. This hybrid is then heat-treated, and dissociation (melting) of the hybrid into single-stranded nucleic acids accompanying the temperature rise is detected by measuring signals such as absorbances, thereby determining the Tm value. Then, based on this Tm value, the mutation is determined. The Tm value becomes higher as the complementarity between the single-stranded nucleic acids composing the hybrid becomes higher, and becomes lower as the complementarity between the same becomes lower. Thus, for example, by using a mutant probe designed so that the target site is a mutant base (X) as the above-described probe, the mutation can be determined in the following manner. First, the Tm value of a hybrid of a target sequence in which the target site is a mutant base with the mutant probe is determined previously (an evaluation standard Tm value). On the other hand, as described above, the Tm value of a hybrid of an amplification product obtained by amplifying the gene with the mutant probe is determined (a measured Tm value). Then, the evaluation standard Tm value and the measured Tm value are compared with each other. As a result, if the measured Tm value is the same as the evaluation standard Tm value, it can be determined that the target sequence of the amplification product shows a perfect match with the probe, i.e., the target site is the mutant base (X), and the mutation is present. On the other hand, if the measured Tm value is lower that the evaluation standard Tm value, the target sequence of the amplification product shows a mismatch with the probe, so that it can be determined that the target site is a normal base (Y), and no mutation is present.
However, the ASP-PCR method has a problem in that it lacks the specificity although it is excellent in sensitivity. For example, when the mutant primer is used, even if no mutation is present at the target site, amplification may be observed, resulting in a false positive. Furthermore, in the ASP-PCR method, only either one of the mutant primer and the normal primer can be used in a single reaction system. Thus, in order to check whether the target site is mutant or normal, it is necessary to perform PCR in two kinds of reaction systems, namely, a reaction system in which the mutant primer is used and a reaction system in which the normal primer is used. Since the two kinds of reaction systems are used as described above, the process thereof is complex and it requires time and costs. On the other hand, the Tm analysis is excellent in specificity, so that the problem of false positives can be avoided. Besides, whether the target site is normal or mutant can be determined in a single reaction system. However, the Tm analysis method has a problem in that it cannot achieve a sufficient sensitivity.
In particular, as described above, when a gene mutation in cancer cells is to be detected, cells having mutant target genes and cells having normal target genes are preset together in a specimen collected from a patient. Thus, for example, it is required to detect the presence or absence of mutation accurately even in a biological sample containing a large amount of normal genes and a small amount of mutant genes.